AOAC 2018 Methods

D. Sample Preparation 1. Use appropriate sterile diluents (Butterfield’s Phosphate Buffer Diluent (BPBD) or Peptone Salt Solution (PPS)). Do not use diluents containing citrate, bisulfate, or thiosulfate with the 3M Petrifilm Rapid E. coli/ Coliform Count Plates, as they can inhibit growth. 2. For food samples, prepare test portion (10, 11, or 50g) or equivalent ratio of sample to diluent to create a 1:10 dilution as appropriate to the sample being tested. 3. For environmental surface samples, add 25 mL of the appropriate diluent to each sponge sample. 4. Blend or homogenize sample as appropriate. 5. For optimal growth and recovery of microorganisms in acidic products (< pH 5), adjust the pH of the sample suspension to greater than pH 5. For acidic products, adjust with pH 1N NaOH. 6. Remove all required plates and allow to come to ambient temperature (20-25 o C). 7. Retract the top film to fully expose the culture medium and dispense 1.0 mL of sample onto the center of the plate. 8. Reapply the cover by rolling down the film. Place the 3M Petrifilm Flat Spreader on the center of the plate and press gently to allow sample to spread evenly over the medium causing gel to form. Let the plate sit undisturbed for at least 1 minute. 9. Incubate the plates a) For dairy products: 30 ± 1 o C or 32 ± 1 o C for coliforms and E. Coli or 42 ± 1 o C for E. coli for 18-24 hours. b) for all other foods: 35 ± 1 o C, 37 ± 1 o C or for coliforms and E. Coli or 42 ± 1 o C for E. coli for 18-24 hours. 10. Enumerate all blue to blue-green colonies with or without gas regardless of size or intensity of color as E. coli. 11 . Interpretation of non- E. coli coliform colonies varies by reference method. a. The United States Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 4 defines coliforms as Gram negative rods, which produce acid and gas from lactose during metabolic fermentation. Enumerate red colonies which are closely associated with entrapped gas within one colony diameter of the colony. Colonies not associated with gas (a distance greater than one colony diameter between colony and gas bubble) are not counted as coliforms. The total coliform count consists of both the red colonies with gas and blue colonies with and without gas, estimates can be made on plates containing greater than 100 colonies. b. ISO defines coliforms by their ability to grow in method-specific, selective media. ISO 48324 enumerates typical coliform colonies on Violet Red Bile Lactose (VRBL) agar, with confirmation of atypical colonies. Enumerate red colonies with and without gas production. The total coliform count is indicated by red colonies with or without gas

3